# Molecular Responses to Climate Change: How Warming and Acidification Reshape the Proteome and Phosphoproteome of the Endangered Mira Chub

## TL;DR

This study explores how warming and acidification affect the protein and phosphorylation responses of the endangered Mira chub, revealing key patterns and stress markers for conservation.

## Contribution

The first proteome and phosphoproteome study of the Mira chub under warming and acidification, identifying novel buffering mechanisms and stress markers.

## Key findings

- Muscle tissue showed more phosphorylation changes than gills, especially under warming.
- Combined stressors triggered a buffering effect, reducing protein-level changes.
- Core biological functions like metabolism and immunity were consistently impacted across stress scenarios.

## Abstract

Global environmental change affects organisms, including their physiology. In freshwater ecosystems, where migration is limited, populations often rely on phenotypic plasticity to respond. While transcriptomics has been widely used to study stress responses at the molecular level, less is known about the proteome, which reflects post‐transcriptional and post‐translational regulation that shapes the resulting phenotype. We conducted the first proteome‐level study on the endangered Mira chub, Squalius torgalensis, which inhabits unstable habitats, enduring harsh summers with high temperatures and frequent droughts. We assessed the effect of warming and acidification, independently and combined, on protein expression and phosphorylation in gills and muscle using tandem mass tags labelling proteomics. While both tissues exhibited similar numbers of differentially expressed proteins, the muscle showed more differentially phosphorylated proteins, particularly under warming. We observed four protein differential expression patterns: consistent regulation across all scenarios, opposite response in one scenario, stress prioritisation in response to dominant stressor (warming), and reduced expression in combined compared to single stressors. The latter suggests a buffering mechanism that limits protein‐level changes under simultaneous stressors, possibly as an energy‐saving mechanism or a consequence of stress overload. A gene set enrichment‐like analysis revealed that, despite the presence of distinct regulatory patterns in each tissue and condition, key biological functions like metabolism, gene/protein expression, and immunity were affected by all stressors. Gene/protein expression was the most affected at the phosphoproteome level. Our findings highlight the importance of proteomics and phosphoproteomics studies to understand species' molecular responses to climate change. By identifying key proteins involved in resilience, we pinpointed candidate stress markers for the Mira chub that can be used to assess the impact of environmental changes. Integrating these tools with genomics and ecological modelling could help improve predictive models for climate adaptation and species conservation.

We conducted the first study focusing on the proteome and phosphoproteome of the endangered Mira chub (Squalius torgalensis) to evaluate its response to warming and acidification. Our analysis revealed four distinct patterns of protein expression, including a buffering effect when subjected to combined stressors: core biological functions, such as metabolism, gene/protein expression, and immune responses. We were able to identify potential stress markers underscoring the importance of proteomics in predicting molecular resilience and pinpointing stress indicators valuable for conservation efforts.